The long term goal is to be able to correctly predict from its sequence (1) how an RNA folds into its biologically functional form, (2) how its structure affects its function, and (3) how fast it folds and responds to proteins and other ligands in its environment. RNA molecules are crucial in all aspects of gene expression. Messenger RNAs transfer the genetic information from DNA to proteins; in RNA viruses the RNA is both the genetic information, and the messenger for protein synthesis. Non-coding RNAs, including microRNAs and small interfering RNAs, regulate transcription of DNA to RNA and translation of RNA to protein. Errors in processing and control of messenger RNAs are linked to human diseases, including neurodegenerative diseases such as Lou Gehrig's disease. RNAs are being investigated as inhibitors of messenger RNAs to silence specific genes, and to cure diseases. RNAs - in humans, viruses, and bacteria - are also outstanding targets for drugs to prevent or cure human diseases. Knowledge of the three-dimensional structures of RNAs, their stabilities, and their rates of interconversion is crucial to understanding all the different biological functions of RNA. In order to obtain this knowledge, newly discovered single-molecule methods are applied: Laser Tweezers and Fluorescence Resonance Energy Transfer (FRET). RNA molecules are synthesized in the laboratory by RNA polymerase from a DNA template. A specific label is attached to each RNA molecule so that it can be monitored and characterized. Laser Tweezers: A micron-sized bead is attached to each end of a single RNA molecule; one bead is held in a micropipette, the other in a laser trap. As the RNA molecule moves, or changes shape as it interacts with other RNAs or proteins, the beads move. Their motion can be seen in a microscope and thus the behavior of the RNA is assessed. FRET: Fluorescent dyes are placed on the RNA or on the molecules it interacts with. The fluorescent signals from the dyes are exquisitely sensitive to the distance between the dyes, and thus on the motion and shape of the RNA. These data provide the thermodynamic stability of the RNA, and its rates of unfolding and refolding. The effects of proteins that unwind, transcribe, and translate the RNA, as well as drugs that inhibit these processes are determined. This information will lead to improved understanding of RNA structure, stability, and dynamics. It will help in understanding RNA function, and in controlling the role of RNA in human diseases.